1. Field of the Invention
The present invention concerns the rapid detection and characterization of viable microorganisms based upon the technique of induced fluorescence. In particular, the present invention concerns employing a non-fluorescent dye substrate for microorganisms to metabolize extracellularly through an enzyme-specific reaction. A specific radiation source can be used to induce product fluorescence and the latter is detected by a conventional photomultiplier tube. Standard curves can be obtained by relating the effect of the microorganism activity to the initial rate of fluorescence generation in order to obtain the identification and estimation of the microorganism concentration.
2. Prior Art
A methodology for rapid detection and identification of microorganisms has long been a concern to the medical, pharmaceutical and food processing fields, among others. Because of this sustained interest, significant advances over the classical time consuming methods of plate counting, membrane filtration, or multiple tube fermentation procedures have been noted. However, these techniques require as much as one to three days in order to detect and identify microorganisms.
Various recent developments have been focused upon techniques in immunofluorescence, enzyme amplification, excitation-emission matrices with a video fluorometer of whole-cell supernatants and differential dye-cell wall binding, mass spectrometry, bacteriophage lysis, computer assisted probability methods, gel ferrography, and the double fluorescent DNA staining method coupled with flow cytometry. Each of these techniques is limited by disadvantages such as laborious and time-consuming laboratory preparation and sample handling, long observation times and nonspecificity with respect to microorganism characterization and subsequent identification. The following patents exemplify some of these prior art procedures.
U.S. Pat. No. 4,283,490 to Plakas discloses a method for detecting the concentration of certain reactive molecules from microbial cells in samples of fluids and degraded solids. A sample containing microbial cells is treated to eliminate substantially all non-microbial materials. The microbial cells are then caused to rupture and form a thin film positioned in the vicinity of a photodetector. An appropriate reagent contacts the thin film and the resulting photon emission is observed by the photodetector through an optical filter which selectively restricts the wave length of photons permitted to pass to the detector. The strength of the luminescence indicates the concentration of microbial cells in the sample.
French Patent 2,504,679 to Koumura, et al discloses a method of detecting microorganisms in a sample solution using a fluorescent analysis method with an umbelliferone derivative. The method consists of: (a) incubating an aqueous solution containing the sample solution and a non-fluorescent umbelliferone derivative at a temperature between 20.degree. and 50.degree. C. until the umbelliferone product is generated in the solution by microorganisms contained in the sample solution; (b) measuring the amount of umbelliferone product generated through induced fluorescence; and (c) determining the concentration of microorganisms in the sample solution from the amount of umbelliferone product generated. In this procedure, the non-fluorescent umbelliferone derivative is hydrolyzed and the umbelliferone product is generated by the microorganisms contained in the sample solution. The product can then be detected by induced fluorescence. The amount of umbelliferone product is approximately proportional to the number of microorganisms. According to the procedure, the microbial inspection is accomplished in one to twelve hours. Generally, it appears that the procedure is most often accomplished in six to twelve hours as evidenced by the examples set forth therein. Moreover, the length of incubation, which is the first step of the procedure, ranges from one to twelve hours itself. Accordingly, although the French patent may describe a procedure which is quicker than known prior art procedures, six to twelve hours for detecting microorganisms remains an unacceptable length of time and thus there remains a need for a quicker procedure.